Validation and depth evaluation of recurrent neural network‐based ultra low‐pass genome sequencing for the detection of absence of heterozygosity: A multi‐centre study of 409 cases

We conducted a comprehensive clinical assessment of our newly developed method, CNVseq-AOH, for the detection of absence of heterozygosity (AOH) using low-pass genome sequencing (LP GS) with ultra-low sequencing data in this multi-centre study.

using LP GS with ultra-low sequencing data.Compared to CMA, CNVseq-AOH showed high sensitivity and great potential to improve the genetic testing of AOH.
Second, to assess and comprehensively study the performance of CNVseq-AOH in a real clinical environment, a total of 409 archived DNA samples of amniotic fluid (209 with positive AOH results (Table S1) and 200 samples with negative AOH results by CMA (Table S3)) from Women's Hospital, Zhejiang University School of Medicine, Jiaxing Maternity and Child Health Care Hospital and Anhui Province Maternity & Child Health Hospital from April 2017 to March 2023, were recruited.Among the 409 samples, CMA identified 209 cases with positive AOHs (506 AOH regions) (Figure 1).LP GS for the 409 samples were conducted on the MGISEQ-2000 platform for single-end (35 bp) sequencing as previously described. 8For these samples, an average of 71.48 M uniquely aligned high quality reads (UAHRs) were obtained, approximately 0.83-fold for each sample.After sequencing, AOH detection was performed for each sample using CNVseq-AOH.
The results of CMA were blinded to individuals who were analysing using CNVseq-AOH.When using the CMA results as a reference, the diagnostic yield of CNVseq-AOH was found to be equivalent to that of CMA (Table S1).Specifically, CNVseq-AOH demonstrated a sensitivity of 100% (209/209) and a specificity of 100% (200/200) in our cohort.
The overlap for the 506 AOH regions detected by CMA and CNVseq-AOH was further analysed (Table S1).It showed that ∼99.60% (504/506) of the AOHs detected by CNVseq-AOH had a reciprocal overlap of more than 50% with the AOHs detected by CMA (Table S2).Compared with CMA, two AOHs were detected to be with an overlap of less than 50% in case PS201 (Figure 2) and case PS44 (Figure 3).Case PS201 included 1 positive AOH across the whole chromosome 9 by CMA.In the CNVseq-AOH detection results, the region was divided into multiple subregions (Figure 2C), with a total overlap of less than 50% between the two methods.Further analysis revealed low-level mosaicism (∼6.7%) of the whole chromosome 9 (Figure 2E).The presence of this mosaicism can affect the performance of CNVseq-AOH, leading to the discrepancy observed between the two methods.For the ∼10.4Mb AOH in case PS44, abnormal signals were detected in the reported regions of CMA (Figure 3C), with some regions showing relatively dispersed signals from allele difference analysis (Figure 3F).These differences may be due to the differences in detection principles between the two methods.
Third, to examine how the detection sensitivity of CNVseq-AOH is affected by sequencing depth, depth evaluation was performed (Figure 1B).In total, the UAHRs for 504 AOHs were utilized to create downsampling samples (14 different sequencing depths for each sample).As a result, the performance of CNVseq-AOH in downsampling samples varied depending on the size of the AOH (Figure 4 and Table S2).The detection sensitivity of 4 AOHs (cases PS1-4) with sizes less than 5 Mb was greatly influenced by UAHR.The detection sensitivity of AOHs with sizes between 5 and 10 Mb was also influenced by UAHR (Figure 4) and reached a plateau at 20 M UAHR.For AOHs larger than 10 Mb and at the chromosome level, the impact of sequencing depth becomes less pronounced (Figure 4 and Table S2), reaching 99.74% and 99.89% at 10 M UAHR.Overall, the detection sensitivity tended to increase as the number of UAHRs increased, and it reached a plateau at 15 M UAHRs for all the 504 AOHs (Figure 4).When using 15 M UAHRs, the overall detection sensitivity is over 99.66%.Therefore, 15 M UAHRs were considered optimal for detecting AOHs using CNVseq-AOH based on our cohort, approximately 23 times (342.86M reads) less than the existing method. 9at is more, CNVseq-AOH addressed the longstanding challenge of standard LP GS in detecting AOH.Typically, the cost of CMA for one sample is more than $600. 10In our laboratory, the overall cost of LP GS for a single case was approximately $248. 8Combining CNVseq-AOH, LP GS could achieve the same level of performance as CMA for AOH detection (> 10 Mb) at a very low sequencing depth.
In summary, we developed a method for predicting AOH using LP GS data and tested its performance in this and Kai Yan designed and performed the experiments.Yan Sun, Zhonghua Wang, Fei Tang, Jianfen Man, Lina Wang, Cangcang Jia, Ping Tang, Xinyi Zhu, Chaohong Wang, Junxiang Tang, Yuanyuan Xia, Xueqin Guo, Kang Zhang and Xiaoli Wang performed data analysis.Yeqing Qian, Lijie Song, Minyue Dong and Yan Sun contributed to revising the manuscript.All authors reviewed the manuscript and approved the submitted version.

F I G U R E 1
Study design.(A) Study workflow; (B) Depth evaluation workflow; (C) The pie chart of 506 absence of heterozygosity (AOH) regions detected in 209 samples with positive chromosomal microarray analysis (CMA) results, including four AOHs < 5 Mb, 21 AOHs with a size between 5 and 10 M, 397 AOHs > 10 Mb and 84 chromosomal level AOHs.

F I G U R E 2
Whole chromosome 9 results for case PS201.(A) Chromosomal microarray analysis (CMA) results; (B) Log-likelihood ratio for haploid and diploid in each bin; (C) Absence of heterozygosity (AOH) prediction likelihoods of CNVseq-AOH; (D) Diagram of chromosome 9; (E) Copy ratio for chromosome 9.

F I G U R E 3
Chromosome 3 results for case PS44.(A) Chromosomal microarray analysis (CMA) results; (B) Log-likelihood ratio for haploid and diploid in each bin; (C) Absence of heterozygosity (AOH) prediction likelihoods of CNVseq-AOH; (D) Diagram of chromosome 3; (E) Copy ratio for chromosome 3; (F) Detailed allele difference in the reported region of arr[GRCh38]3p22.1p21.1(4317528153596971)x2 hmz by CMA.multi-centre study.CNVseq-AOH can identify regions of AOH accurately in the clinical setting and possesses great potential to improve the genetic testing of AOH.According to these findings, CNVseq-AOH has demonstrated high precision in detecting AOHs, indicating its promising application in clinical settings.A U T H O R C O N T R I B U T I O N S Suping Li, Lijie Song, Jiansheng Zhu, Minyue Dong, Yeqing Qian, Jianjun Zhu, Zhiguo Tang and Yan Sun contributed to the conception and design of the study.Yan Sun wrote the first draft of the article.Yun Yang, Linlin Fan, Yixi Sun, Bei Liu, Min Chen, Yuqin Luo, Junjie Hu F I G U R E 4 Depth evaluation.Detection sensitivity of CNVseq-AOH for downsampling samples.The dotted red line shows the optimal uniquely aligned high quality reads (UAHR) (15 M).